Neuroblastoma cell lines expressing the alpha2delta subunit of calcium channels and methods therefore

ABSTRACT

The present invention provides methods for identifying test substances that bind to the α2δ subunit of a calcium channel.

FIELD OF THE INVENTION

[0001] The present invention relates to neuroblastoma cells that expressthe α2δ subunit of the calcium channel. In another aspect of thisinvention, the invention relates to methods and assays usingneuroblastoma cells and neuroblastoma cell membranes. The cells of thepresent invention are useful for discovering new compounds that modulatethe function of the α2δ subunit of calcium channel.

BACKGROUND OF THE INVENTION

[0002] Several subtypes of the α2δ subunit of calcium channel have beencloned (Angeloni et al. Mol. Cell. Probes 14:53-54, 2000; Gao et al., J.Biol. Chem. 275:12237-12242, 2000; and PCT Application WO 99/23519).

[0003] Neuroblastoma cell membranes contain N-type calcium channels andneuroblastoma cells have been used as a model for neuronaldifferentiation (Bruhn, et al. Endocrinology 137:572-9, 1996; Gotti, etal. Differentiation (Berlin) 34:144-55, 1987; Hogg et al. Pharmacol.,312:257-261, 1996; and Kurata, et al. FEBS Lett., 321:201-4, 1993).5-Bromo-2′-deoxyuridine (BrdU) induces morphological and functionaldifferentiation of neuroblastoma cells, resulting in an increase ofneurotransmitter receptors and the release of neurotransmitters(Clementi, et al. Adv. Exp. Med. Biol. 296:91-102, 1991).[¹²⁵I]co-conotoxin binding sites were increased in the differentiatedneuroblastoma IMR32 cells, indicating that the N-type calcium channelswere increased in the cells (Carbone et al., Pfluegers Arch., 416:170-9,1990). Recently, Western blot analysis has shown that the β1b subunit ofcalcium channels is the predominant isoform expressed in IMR32 cells(McEnery, et al. FEBS Lett. 420: 74-78, 1997).

[0004] Gabapentin (GBP) is an anticonvulsant that has shown usefulnessin the treatment of neuropathic pain (Backonjy, M. et al. J. Am. Med.Assoc. 280:1831-1836, 1998; Laird and Gidal, Ann. Pharmacother.34:802-807, 2000; and Rowbotham et al. M., J. Am. Med. Assoc.,280:1837-1842, 1998). GBP inhibits neurotransmitter release (Dooley, etal. Neurosci. Lett. 280:107-110, 2000) and inhibits calcium currents inbrain neurons (Fink, et al. Br. J. Pharmacol. 130:900-906, 2000; Laridand Gidal, supra; and Stefani et al. Neuropharmacology 37:83-91, 1998).Interestingly, a high-affinity binding site for GBP was found in braintissue and the target protein was identified as the α2δ subunit ofsubunit of calcium channels (Brown and Gee, J. Biol. Chem.,273:25458-25465, 1998; Dissanayake, et al. Br. J. Pharmacol.,120:833-840, 1997; and Gee et al., J. Biol. Chem. 271:5768-76, 1996). Anautoradiographic binding study showed that the GBP binding site waswidely distributed in rat brain areas such as frontal cortex, striatum,hippocampus and cerebellum (Hill and Woodruff, Eur. J. Pharmacol. Mol.Pharmacol. Sect., 244:303-9, 1993; Thurlow et al. Br. J. Pharmacol.,118;457-465, 1996). Therefore compounds that compete with Gabapentinbinding to the α2δ subunit should be useful as anticonvulsants and inthe treatment of neuropathic and chronic pain.

SUMMARY OF THE INVENTION

[0005] The present invention relates to a method for detecting bindingof a test substance to an α2δ subunit comprising the steps of:contacting a neuroblastoma cell membrane sample comprising the α2δsubunit with gabapentin and a test substance; detecting binding ofgabapentin to the cell membrane; and, comparing the level of binding ofgabapentin as compared with a control sample lacking the test substance.

[0006] In one embodiment the cell membranes are part of intact cells andin another embodiment the cell membranes are obtained from isolated cellmembrane preparations. In one embodiment the cells are the neuroblastomacells IMR32. Preferably the neuroblastoma cell membranes aredifferentiated neuroblastoma cell membranes and in one embodiment thedifferentiated cell membranes are obtained following cell incubationwith BrdU.

[0007] In a preferred method, the methods of this invention furthercomprise the step of separating the cell membranes from unboundgabapentin. In another preferred method, the comparing step comprisesmeasuring binding of labeled gabapentin bound to the cell membranes. Theinvention further relates to compounds identified using the methods ofthis invention.

[0008] In another embodiment the invention further relates to a testsubstance identified by a method comprising the steps of: contacting aneuroblastoma cell membrane sample comprising the α2δ subunit of acalcium channel with gabapentin and a test substance; detecting bindingof the gabapentin to the cell membrane; and comparing the level ofbinding of gabapentin as compared with a control sample lacking the testsubstance.

[0009] The invention further relates to a method for identifying a testsubstance capable of binding to an α2δ subunit of a calcium channelcomprising the steps of: incubating an IMR32 cell membrane withradioactive gabapentin (GBP) and a test substance, wherein the membranecomprises an α2δ subunit of calcium channel and where the contact is forsufficient time to allow GBP binding to the α2δ subunit of calciumchannels in the cell membranes; separating the cell membranes fromunbound radioactive GBP; measuring binding of the radioactive GBP to thecell membranes; and identifying a compound that inhibits GBP binding bya reduction of the amount of radioactive GBP in the measuring step to anestablished control.

[0010] The methods of the present invention are useful for identifyingcompounds that interact with the α2δ subunit of calcium channels.Compounds identified using the methods of the present invention can thenbe tested for their ability to treat biological conditions mediated bythe α2δ subunit of calcium channels.

BRIEF DESCRIPTION OF THE DRAWINGS

[0011]FIG. 1. Effect of membrane protein concentration on [³H]GBPbinding. The membranes of human neuroblastoma IMR32 cells were incubatedwith [³H]GBP (10 nM) at 25° C. for 60 min. The data are representativeof two experiments with each point assayed in duplicate. Blacksquares=Total bound counts; Clear squares=Specific binding; Cleartriangles =nonspecific binding.

[0012]FIG. 2. Saturation binding of [³H]GBP to IRM32 cell membranes. (A)Varying concentrations of [³H]GBP were incubated with membranes (40 μgprotein/ml) from non-differentiated IMR32 cells. Black squares=Totalbound counts; Black circles =Specific binding; Clear squares=nonspecific binding.

[0013] (B) Varying concentrations of [³H]GBP were incubated withmembranes (40 μg protein/ml) from IMR32 cells differentiated by 10 μMBrdU. Black squares=Total bound counts; Black circles=Specific binding;Clear squares=nonspecific binding.

[0014] (C) Scatchard plot from the data of FIG. 2A and FIG. 2B. Clearsquares=differentiated IMR32 cells; Black squares=nondifferentiatedIMR32 cells

[0015]FIG. 3. Inhibition of [³H]GBP binding to human neuroblastoma IMR32cell membranes by GBP and L-methionine. Varying concentrations ofunlabeled GBP and L-methionine were incubated with membranes (20 μgprotein/ml) of non-differentiated and differentiated IMR32 cells in thepresence of 10 nM [³H]GBP. The results shown represent two experimentswith each point assayed in duplicate. Black squares=GBP binding tonondifferentiated IMR32 cells; Black triangles=L-methionine binding tonondifferentiated IMR32 cells; Clear squares=GBP binding todifferentiated IMR32 cells; Clear triangles=L-methionine binding todifferentiated IMR32 cells

DETAILED DESCRIPTION OF THE INVENTION

[0016] The present invention provides methods to identify compounds thatmodulate the function of the α2δ subunit of the calcium channel inneuroblastoma cells and preferably in differentiated neuroblastomacells.

[0017] There are a variety of neuroblastoma primary cells and cell linesthat can be used in the methods of this invention. For purposes of thisinvention the term “neuroblastoma cell lines” includes cell lines, cellsisolated from tumor explants and cell hybrids prepared by fusion of acell with a neuroblastoma cells. Preferred neuroblastoma cells of thepresent invention are of human origin but can also be of animal origin.Exemplary cells and cells lines include, but are not limited to, IMR32cells, SK-N-MC cells and NG 108 cells (a mouse neuroblastoma/rat gliomahybrid cell line). These cells are available from a number of sourcesincluding the ATCC (Manassas, Va.). While established cell lines arepreferred, cells useful for this invention can also be isolated from avariety of vertebrate sources, such as animal or human tumor explants.

[0018] In a preferred embodiment, IMR32 cells are used and in anotherpreferred embodiment the neuroblastoma cells are exposed to adifferentiation-promoting agent, such as BrdU. Otherdifferention-promoting agents that can be used in this inventioninclude, but are not limited to, dibutryl cyclic AMP, neural growthfactor (NGF) and retinoic acid. Differentiated neuroblastoma cells tendto express increased levels of α2δ subunit protein as compared withundifferentiated cells.

[0019] There are a number of methods known in the literature fordifferentiating undifferentiated neuronal cells, such as neuroblastomacells, and an exemplary method is found in the example section, providedbelow. In general, however, the cells are exposed to a non-cytotoxicamount of the differentiation-promoting agent for a time sufficient toinduce differentiation in the cell culture. Differentiation can bedetermined visually, through the outgrowth of neural processes, forexample, or in the expression of a more differentiated phenotype,including increased adherence to the substrate, improved growth controland a general flattening of the cell morphology. Alternatively, celldifferentiation can be determined by detecting proteins that are knownto be associated with a more differentiated cell phenotype, as is knownin the art.

[0020] The assays of this invention can be used as intact cell assays orthe assays can be performed using membrane lysates or purified membranepreparations from cells expressing the calcium channel α2δ subunit.Where cells membranes are used, the α2δ subunit of calcium channels isisolated as a component of the neuroblastoma cell membranes. Isolatedcell membranes are prepared using conventional means such ashomogenizing the cells via mechanical force.

[0021] Gabapentin (GBP) (CAS number 60142-96-3) is used as a specificligand for the α2δ subunit of calcium channels. In a preferredembodiment the GBP is labeled to facilitate detecting GBP binding to theα2δ subunit. GBP can be labeled using any number of methods known in theart including fluorescent labels, radioactive labels, and the like. In apreferred embodiment, a commercially available [³H]GBP is used as adetectable ligand in the displacement assays of the present invention.

[0022] In the assays of the present invention, the cell membranes arecombined with GBP and a test substance. The test substance can be anycandidate molecule that one hypothesizes will bind to the α2δ subunit.These include small molecules, peptides, polypeptides, includingantibodies, and the like.

[0023] The cell membrane mixture comprising GBP and the test substanceare then incubated in an aqueous buffer for a time sufficient to permitthe GBP to bind to the α2δ subunit contained in the cell membranes. Theamount of incubation time necessary depends on the amount of reagentsused, temperature, and other factors. Varying the reaction conditionsusing methods well known in the art alters the amount of labelincorporation into the cell membranes and these conditions can bereadily optimized by those of ordinary skill in the art. Afterincubation the cell membranes are isolated from unbound GBP usingconventional means, including filtration or centrifugation.

[0024] The ability of the test substance to bind to the α2δ subunit isdetermined by measuring a reduction in the amount of GBP binding to thecell membranes in samples containing both GBP and test substance ascompared to a control reaction that does not include the test substanceand which preferably includes both unlabeled and labeled GBP. Where theGBP is radiolabeled, the level of radioactivity in treated cellmembranes compared to control membranes is measured.

[0025] Candidate compounds that are capable of competing with GBP forbinding are identified using the methods of the present invention. Thesecompounds can then be tested for their ability to affect a number ofbiological conditions mediated by the α2δ subunit of calcium channels.

[0026] The present invention is exemplified by way of the followingexamples. These examples are not intended to limit the presentinvention.

EXAMPLE 1 Differentiation of IMR32 Cells and Analysis of the Expressionof the α2δ Subunit of Calcium Channels

[0027] Materials and Methods

[0028] IMR32 cells, SK-N-MC cells and NG 108 cells as well as cellstreated in culture with 10 μM of BrdU for 10 to 12 days were used. Cellswere harvested and homogenates were made in HEPES/KOH buffer, pH 7.4.The homogenates were centrifuged for 15 min at 1000× g, and thesupernatants were centrifuged at 40000× g for 15 min to obtain cellmembranes.

[0029] A [³H]GBP binding assay was performed as described by Gee, et al.(supra). Non-specific binding was defined in the presence of 100 μMunlabeled GBP. Separation of bound from free ligand was effected byfiltration through 0.3% polyethylenimine-soaked GF/B filters. Thefilters were washed with 3×4 ml of 10 mM HEPES pH 7.4. Radioactivity onfilters was determined by scintillation counter.

[0030] [³H]GBP binding increased with increasing concentrations of cellmembranes. [³H]GBP bound to cell membranes from all neuroblastoma celllines tested. The increase in binding was linear with membraneconcentrations up to 100 μg protein /ml (FIG. 1).

[0031] In one example to test the effect of BrdU on the density of[³H]GBP binding sites in the differentiated 1MR32 cells, saturationexperiments were performed using both undifferentiated anddifferentiated 1MR32 cell membranes (FIG. 2). Scatchard analysis of thesaturation binding data suggests a single high affinity GBP binding siteon IMR 32 cell membranes with a K_(d) value of 37 nM and Bmax value of1186 fmol/mg protein. BrdU increased the expression of [³H]GBP bindingsites (B_(max) =2245 fmol/mg protein) without changing its affinity(K_(d)=39 nM).

[0032] The pharmacology of [³H]GBP binding sites was examined byinvestigating the ability of unlabeled GBP and L-methionine to inhibitbinding. 10 nM of [³H]GBP was used for the experiments. The K_(i) valueswere obtained using a one-site binding model and are summarized inTable 1. FIG. 3 shows that unlabeled GBP and L-methioninedose-dependently inhibited [³H]GBP binding to IMR32 cell membranes. BrdUdid not significantly alter the K_(i) values of GBP and L-methionine forthe cell membranes (Table 1). TABLE 1 K_(i) values of GBP andL-methionine inhibition of [³H]GBP binding to undifferentiated (Control)and differentiated (BrdU) IMR32 cell membranes. K_(i) (nM) Control BrdUGBP 29 15 L-methionine 39 30

[0033] The increase in [³1H]GBP binding by BrdU in neuroblastoma cellssuggests that the α2δ subunit of calcium channels increases during thedifferentiation of human neuroblastoma cells. This increase, in additionto previous results, which indicated that the α1 and β1b subunits werealso increased by BrdU (Carbone, et al., supra and McEnery et al.,supra), suggests that most of the major subunits of calcium channels areup-regulated during the differentiation of human neuroblastoma cells.Full activation of calcium channels requires both β and α2δ subunits(Shistik, et al. J. Physiol. (London):489:55-62, 1995 and Walker andDeWaard, Trends Neurosci., 21:148-154, 1998). The present results implythat calcium channels play an important role in the differentiation ofhuman neuroblastoma cells.

[0034] Several L-amino acids, such as leucine, methionine, phenylalanineand valine, inhibit [3H]GBP binding to synaptic plasma membranes.L-methionine is one of the most potent inhibitors (Thurlow, et al., Eur.J Pharmacol. Mol. Pharmacol. Sect., 247:341-5, 1993). We found that GBPand L-methionine inhibited [³H]GBP binding to IMR32 cell membranes. TheK_(i) values of GBP and L-methionine were not significantly differentbetween undifferentiated and differentiated cell membranes, indicatingthat BrdU did not change the affinities of GBP and L-methionine for cellmembrane binding.

[0035] GBP binding to calcium channels requires both α2 and δ subunits(Wang, et al. Biochem. J, 342:313-320, 1999). The region between theN-terminal end and the first transmembrane domain of α2, as well as theregion between the splicing acceptor sites may play important roles inmaintaining the structural integrity for GBP binding.

What is claimed is:
 1. A method for detecting binding of a testsubstance to an α2δ subunit of a calcium channel comprising the stepsof: (a) contacting a neuroblastoma cell membrane sample comprising theα2δ subunit of a calcium channel with gabapentin and a test substance;(b) detecting binding of the gabapentin to the cell membrane; and (c)comparing the level of binding of gabapentin as compared with a controlsample lacking the test substance.
 2. The method of claim 1 wherein thecell membranes are part of intact cells.
 3. The method of claim 1wherein the cell membranes are obtained from an isolated cell membranepreparation.
 4. The method of claim 1 wherein the neuroblastoma cellmembranes are IMR32, SK-N-MC or NG 108 cell membranes.
 5. The method ofclaim 1 wherein the neuroblastoma cell membranes are differentiatedneuroblastoma cell membranes.
 6. The method of claim 5 wherein thedifferentiated cell membranes are obtained following incubation withBrdU.
 7. The method of claim 1 further comprising the step of separatingthe cell membranes from unbound gabapentin.
 8. The method of claim 1wherein the comparing step comprises measuring binding of labeledgabapentin bound to the cell membranes.
 9. A compound identified usingthe method of claim
 1. 10. A test substance identified by a methodcomprising the steps of: (a) contacting a neuroblastoma cell membranesample comprising the α2δ subunit of a calcium channel with gabapentinand a test substance; (b) detecting binding of the gabapentin to thecell membrane; and (c) comparing the level of binding of gabapentin ascompared with a control sample lacking the test substance.
 11. A methodfor identifying a test substance capable of binding to an α2δ subunit ofa calcium channel comprising the steps of: (a) incubating an IMR32 cellmembrane with radioactive gabapentin (GBP) and a test substance, whereinthe membrane comprises an α2δ subunit of calcium channel and where thecontact is for sufficient time to allow GBP binding to the α2δ subunitof calcium channels in the cell membranes; (b) separating the cellmembranes from unbound radioactive GBP; (c) measuring binding of theradioactive GBP to the cell membranes; and (d) identifying a compoundthat inhibits GBP binding by a reduction of the amount of radioactiveGBP in step (c) to an established control.